SYBR green: melt curve Tm shift
(by ebs15242 from creighton.edu)
Mon Dec 21 10:57:39 EST 2009
I am having a small problem with my SYBR green qrtpcr.
I have 4 primer sets running against 2 different cDNA
samples. The primers are all at the same concentration
(300 nm). I prepared both cDNA samples side by side, in
exactly the same way and loaded 10ng of each.
The problem is, when I look at the melt curve, I'm seeing
a decrease in Tm of the product of about 1 degree C from
one cDNA to another. This Tm shift occurs for all 4 primer
sets. In both cases, there is a single sharp peak, so I'm
doubting I have primer dimers.
Does anyone have any ideas? I've used these primers before
on different samples, and the melting curves always exactly
coincided, no matter what the cDNA was.
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